Phycobiliprotein Evolution (Phycoerythrin/Phycobilisomes/Cell Wall/Photosynthesis/Prokaryotic Evolution) THOMAS A
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Proc. Natd Acad. Sci. USA Vol. 78, No. 11, pp. 6888-6892, November 1981 Botany Morphology of a novel cyanobacterium and characterization of light-harvesting complexes from it: Implications for phycobiliprotein evolution (phycoerythrin/phycobilisomes/cell wall/photosynthesis/prokaryotic evolution) THOMAS A. KURSAR*, HEWSON SwIFTt, AND RANDALL S. ALBERTEt tBarnes Laboratory, Department ofBiology, and *Department of Biophysics and Theoretical Biology, University of Chicago, Chicago, Illinois 60637 Contributed by Hewson Swift, July 2, 1981 ABSTRACT The morphology of the marine cyanobacterium After examining the in vivo spectral properties of several of DC-2 and two light-harvesting complexes from it have been char- the recently discovered species ofcyanobacteria, it came to our acterized. DC-2 has an outer cell wall sheath not previously ob- attention that one of the PE-containing types termed DC-2 served, the purified phycoerythrin shows many unusual proper- showed some rather unusual features. Further study revealed ties that distinguish it from all phycoerythrins characterized to that this species possesses novel PE, phycobilisomes, and outer date, and isolated phycobilisomes have a single absorption band cell wall sheath; these characteristics suggest that it should be at 640 nm in the phycocyanin-allophycocyanin region of the spec- trum. On the basis of these observations we suggest that DC-2, placed in a new phylogenetic branch for the cyanobacteria. rather than being a member of the Synechococcus group, should be placed in its own taxonomic group. In addition, the particular MATERIALS AND METHODS properties of the isolated phycoerythrin suggest that it may be An axenic representative of an early stage in the evolution of the phyco- isolate of Synechococcus sp., clone DC-2, obtained erythrins. These observations are ofspecial interest in light ofthe from R. R. L. Guillard, was grown in f/2 enriched seawater contribution DC-2 and related cyanobacteria may make to global medium (9) at 20°C with aeration. Cells were disrupted in a primary productivity. French pressure cell and cellular debris and membranes were removed by centrifugation. The supernatant was fractionated Several species of blue-green- and red-pigmented marine cy- with ammonium sulfate to purify PE. The 20-40% saturated anobacteria that not only are abundant in the world's oceans but ammonium sulfate cuts were combined and dialyzed against 10 also may be responsible for a significant fraction ofprimary pro- mM sodium phosphate (pH 6.8) containing 0.2 M sodium chlo- ductivity have been discovered recently (1, 2). The red-pig- ride at 5°C. The dialyzed sample was loaded on hydroxylapatite mented cells have the virtue that they are readily distinguished and eluted with increasing concentrations ofsodium phosphate from most other phytoplankton species by their principal in vivo (pH 6.8). The fractions having the highest Au3/A2w were fluorescence emission in the orange (560-580 nm), which un- pooled and rechromatographed as before. This PE fraction was doubtedly arises from the presence ofthe red pigment-protein, passed through a Sephadex G-200 column equilibrated with 50 phycoerythrin (PE). In some cyanobacteria and most red algae, mM sodium phosphate (pH 7.0). The main fraction was eluted PE serves as the major light-harvesting pigment for photosyn- with 50 mM sodium phosphate (pH 7.0), collected and rechro- thesis and is found in association with the other major phycobili- matographed on G-200. The A543/A2w ratio was 8.2 after both pigments, phycocyanin and allophycocyanin. In vivo these phy- the second and third passes through G-200. The final yield of cobilipigments are aggregated into macromolecular arrays that PE was 31%. All chromatographic procedures were conducted form discrete organelles called phycobilisomes and are attached at 20°C. Phycobilisome-like particles were prepared from the to the chlorophyll a-containing photosynthetic lamellae (3). cells as described (10). All spectral characterizations were con- Such an organization allows for excitation energy transfer from ducted at 20°C on either an Aminco DW-2 dual beam spectro- the shortest-wavelength-absorbing pigment, PE, to phycocy- photometer or an SPF-500 corrected spectrum fluorometer. A anin and allophycocyanin and ultimately to the chlorophyll a Cary 60 spectropolarimeter was provided by the Department residing in the photochemical reaction centers ofphotosystems of Chemistry, University of Chicago. The concentration of the I and II (3-5). Therefore, the phycobilipigments serve two im- phycourobilin (PUB) and phycoerythrobilin (PEB) chromo- portant functions in photosynthesis: (i) they increase photon phores were measured in 8 M urea (pH 2.0) containing 10 mM capture under light-limited conditions and (ii) they increase the 2-mercaptoethanol (11). spectral range of light energy available for photosynthesis by Cells were prepared for electron microscopy by fixing in 4% absorbing light where chlorophyll a absorbs weakly and where (wt/vol) glutaraldehyde in ultrafiltered sea water buffered to there is the greatest transmission oflight in the water column. pH 7.4 with 10 mM sodium cacodylate. The cells were postfixed Recent studies have demonstrated that the phycobiliproteins in 1% osmium tetroxide, embedded in Epon, and stained with have highly conserved NH2-terminal sequences (6-8) and that uranyl acetate and lead citrate. phycobilisome structure is also fairly conservative (3). The dual requirements for efficient energy transfer from the phycobili- RESULTS AND DISCUSSION pigments to chlorophyll a and for assembly of a phycobilisome seem to impose strong constraints on the nature ofthe phycobil- With the exception of the outer sheath, the cells of DC-2 re- ipigments and their in vivo macromolecular organization. sembled the related genera Anabaena (12) and Microcystis (13) in structure. Cell profiles were 0.6-1.2 Im in width (average The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: PE, phycoerythrin; PUB, phycourobilin; PEB, phy- nmnt" in accordance with 18 U. S. C. §1734 solely to indicate this fact. coerythrobilin; CD, circular dichroism. 6888 Downloaded by guest on September 29, 2021 Botany: Kursar et aL Proc. NatL Acad. Sci. USA 78 (1981) 6889 0.8), and were up to 4.0 Aum in length (Fig. la). Cell division energy within the pigment-protein to longer wavelength (flu- involved constriction and binary fission as in related genera. A orescing) chromophores at the 565-nm absorption band (20-23). distinctive feature of DC-2 is its outer sheath, composed of a In isolated PE these chromophores will fluoresce, whereas in series ofparallel ridges spaced about 300 A apart, serrate in cross vivo the excitation energy is transferred to an adjacent light- section, branched at ends ofthe cell, and running parallel to its harvesting pigment-protein. long axis (Fig. lb). Sheath components in Anacystis (14) and The 543-nm absorption peak of DC-2 PE assigned to PE is Anabaena (15) lack this complex orientation. Ridges were at- symmetrical and relatively narrow; the bandwidths at halfmax- tached at their bases to a trilaminar extracellular membrane, imum of DC-2 PE and C-PE are 1060 cm-' and 1880 cm-', similar to that described for other cyanobacteria (16). In some respectively. Denaturation ofPEs with detergents, heat, urea, cells (e.g., as in Fig. 1c), cytoplasmic lamellae were closely ap- sulfhydryl reagents, etc. results in the loss of the long-wave- pressed to the inner surface of the cell membrane, producing length absorption band and the appearance of a nearly sym- a multilayered cell margin. metrical maximum at about 540-550 nm (24-29). This repre- The absorption spectrum of the purified PE of DC-2 has sents the absorption maximum of a protein-bound PEB maxima at 543, 497, 375, and 304 nm (Fig. 2). The chromo- molecule for which there is a minimal protein-chromophore phores of PE include PUB and PEB, which are linear tetra- interaction. The 565-nm absorption bands of the PEs are be- pyrroles related to the bile pigment biliverdin. The 497-nm lieved to be due to either protein-chromophore or chromo- band is assigned to PUB and the 375- and 543-nm bands are phore-chromophore interactions (26). Therefore native DC-2 assigned to PEB. The ratio of PEB to PUB in the purified PE PE has an absorption maximum similar to that ofdenatured PEs was determined to be 4:1 (see Materials and Methods). Most and the distinct "fluorescing" PEB found in all other PEs may PEs previously described, including the C-, B-, and R- spec- be (i) absent, (ii) blue-shifted 10-20 nm, or (iii) found only in troscopic types, have a markedly asymmetric absorption spec- the fully aggregated complex. These observations suggest that trum with a maximum at 560-565 nm and a second maximum the PEBs ofDC-2 PE have electronic and vibrational states that or shoulder at 545 nm (7, 17); for example, the R-PE of Gra- are only weakly perturbed by the protein. The fluorescence cilaria (Fig. 2). Two exceptions are the PE ofOscillatoria (Tri- emission spectrum of DC-2 PE has a maximum at 563 nm and chodesmium) thiebauti and PE-I of the cryptomonads, which a shoulder at 605 nm. The main emission band is also blue- have a shoulder rather than a maximum at 550-560 nm (18,19). shifted with respect to other PEs, all of which have emission From an analysis ofthe polarization offluorescence otherwork- peaks at 570-578 nm (Fig. 2; ref. 7). ers have concluded that 545-nm band of C-, B-, or R-PE rep- The visible circular dichroism (CD) spectrum ofDC-2 PE has resents "sensitizing" PEB chromophores that transfer excitation several unique properties (Fig. 3). First, the negative Cotton a b c FIG. 1. Electron micrographs of chroococcalean cyanobacterium DC-2.